The cell division cycle is a tightly controlled multi-gene process. It is characterized by the coordinated replication and segregation of cellular components, culminating in cleavage furrow formation and cytokinesis. Although the cell cycle is generally conserved, trypanosomes have unique features that prevent identification of many regulatory components by direct comparisons with yeast or mammalian systems. The present study seeks to construct tagged marker trypanosomes that can be used in conjunction with high throughput approaches to identify cell cycle regulatory genes. Novel features of the trypanosome cell cycle make it uniquely amenable to these methods. Whereas mammalian cells and yeast generally halt cell cycle progression when a step in the process is disrupted, trypanosomes lack appropriate checkpoint controls. Consequently, cell cycle disruption is accompanied by the accumulation of nuclei, flagella or kinetoplasts, and the inappropriate segregation of basal bodies or Golgi. The present proposal takes advantage of this situation by generating tagged cells labeled in each of the compartments that show significant changes when the cell cycle is disrupted. The tagged cells will be useful to the general research community for the rapid assessment of organelle replication and segregation in response either to drugs, up-regulation of target genes, or the conditional knockdown of genes by RNAi. Here, the cells will be constructed, validated, and used to screen an RNAi library. The project has three specific aims. The first aim is to construct procyclic form (PF) cell lines that constitutively express three compartmentalized fluorescent tags. We have already constructed dually labeled cells with nuclear-GFP and flagellar-DsRed. The second aim is to demonstrate that the tagged cells have normal growth and morphology, and allow rapid assessment of cell cycle defects by microscopy and flow cytometry. The third aim is to use flow cytometry to screen an RNAi library from T. brucei for genes that regulate the cell cycle. Here we will use our fluorescent cells and the cell sorting capabilities of the flow cytometer to segregate mutant cells from a bulk population. Sorting rates up 70,000 cells per second can be achieved. The project is significant at several levels. The tagged cell lines will benefit the research community by providing a tool for rapid assessment of organelle replication and segregation. Currently this assessment is time consuming to perform, and cannot be accomplished without specialized antibody reagents that are not present in most labs. Our use of these cells to screen an RNAi library is anticipated to identify new cell cycle regulatory components. Because the screen selects for genes that are essential for cell proliferation, some of the genes are likely to include rational targets for therapy design. PUBLIC HEALTH RELEVANCE: Human African trypanosomiasis (HAT) is a WHO Category I disease (emerging or out of control). New therapies are sorely needed. Anti-proliferative therapies directed against cell cycle components are becoming increasingly important in the control of some cancers. The current proposal seeks to develop fluorescently tagged trypanosome cell lines as part of a high throughput screen to identify cell cycle genes. Because cell proliferation is essential to the infection cycle, the study is expected to identify new proteins that are sensitive to therapeutic intervention. The fluorescently tagged cell lines will also be useful for other public health studies where a rapid evaluation of organelle replication and segregation is important.